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Abstract

Background

The development of COPD in subjects with alpha-1 antitrypsin (AAT) deficiency is likely
to be influenced by modifier genes. Genome-wide association studies and integrative
genomics approaches in COPD have demonstrated significant associations with SNPs in
the chromosome 15q region that includes CHRNA3 (cholinergic nicotine receptor alpha3) and IREB2 (iron regulatory binding protein 2).

We investigated whether SNPs in the chromosome 15q region would be modifiers for lung
function and COPD in AAT deficiency.

Methods

The current analysis included 378 PIZZ subjects in the AAT Genetic Modifiers Study
and a replication cohort of 458 subjects from the UK AAT Deficiency National Registry.
Nine SNPs in LOC123688, CHRNA3 and IREB2 were selected for genotyping. FEV1 percent of predicted and FEV1/FVC ratio were analyzed as quantitative phenotypes. Family-based association analysis
was performed in the AAT Genetic Modifiers Study. In the replication set, general
linear models were used for quantitative phenotypes and logistic regression models
were used for the presence/absence of emphysema or COPD.

Results

Three SNPs (rs2568494 in IREB2, rs8034191 in LOC123688, and rs1051730 in CHRNA3) were associated with pre-bronchodilator FEV1 percent of predicted in the AAT Genetic Modifiers Study. Two SNPs (rs2568494 and rs1051730)
were associated with the post-bronchodilator FEV1 percent of predicted and pre-bronchodilator FEV1/FVC ratio; SNP-by-gender interactions were observed. In the UK National Registry
dataset, rs2568494 was significantly associated with emphysema in the male subgroup;
significant SNP-by-smoking interactions were observed.

Conclusions

IREB2 and CHRNA3 are potential genetic modifiers of COPD phenotypes in individuals with severe AAT
deficiency and may be sex-specific in their impact.

Keywords:

Background

Chronic obstructive pulmonary disease (COPD) is a complex disease characterized by
persistent airflow limitation. COPD risk likely results from the cumulative effect
of environmental factors (especially cigarette smoking), genetic factors, and gene-by-environment
interactions [1]. Alpha-1 antitrypsin (AAT) deficiency, typically caused by homozygosity for the Z
allele at the AAT gene (SERPINA1), is a proven genetic cause of COPD. However, the development of COPD and emphysema
in subjects with AAT deficiency is highly variable and is likely influenced by modifier
genes and environmental factors [2-4].

Spirometric measurements of pulmonary function are widely used phenotypes in evaluating
AAT deficient subjects and monitoring lung function decline [5]; CT scan assessments for emphysema have been used as additional intermediate phenotypes
of COPD to overcome some of the heterogeneity inherent in spirometric classifications
alone. Familial aggregation studies of pulmonary function have suggested additional
modifier genes in AAT deficiency subjects [6,7]. A few potential AAT candidate modifier genes, including NOS3 [8], GSTP1 [9], TNF [10], and IL10 [11], have been reported so far, but these results have not been consistently replicated.

Genome-wide association (GWA) studies have revolutionized the identification of susceptibility
genes for complex diseases. Three recent GWA studies showed that SNPs in a region
of chromosome 15q25 were significantly associated with lung cancer; several nicotinic
acetylcholine receptor genes, including CHRNA3 and LOC123688, are located in this region [12-14]. A genome-wide association (GWA) study in COPD also showed significant associations
between COPD susceptibility and SNPs in this region [15]. This region was also associated with airflow obstruction and emphysema [16,17]. Interestingly, in addition to nicotinic acetylcholine receptor genes, this region
also includes IREB2 (iron regulatory binding protein 2). IREB2 was identified as a potential COPD susceptibility gene using an integrative genomics
approach with gene expression analysis of lung tissue samples followed by genetic
association analysis [18]. We hypothesized that SNPs in this chromosome 15q region may be modifiers of intermediate
phenotypes of COPD in subjects with severe AAT deficiency.

Methods

Study subjects

The current analysis included 378 subjects with severe AAT deficiency (protease inhibitor
[PI] ZZ) from 167 families in the AAT Genetic Modifiers Study. Ascertainment of eligible
sibling pairs was based on homozygosity for the Z allele at the SERPINA1 locus as previously described [19]. Pre- and post-bronchodilator study spirometry testing was performed according to
American Thoracic Society (ATS) standards as described previously [19]. Percent predicted values for FEV1 were calculated using equations of Crapo and colleagues for Caucasian subjects [20]. The FEV1/FVC ratio was analyzed using unadjusted values. Pack-years of cigarette smoking were
calculated by multiplying the number of years smoked by the average number of daily
cigarettes smoked, divided by 20. All participants provided written informed consent,
and the study protocol was approved by individual Institutional Review Boards at each
of the participating clinical centers (Partners IRB, 2001P001237). 458 unrelated Caucasian
subjects from the UK AATD National Registry were also genotyped. Approval for the
study was given by the local ethics committee. All patients had a serum alpha-1 antitrypsin
(AAT) level of < 11 μM and PI ZZ genotype confirmed by allele-specific PCR (Heredilab,
Salt Lake City, Utah, USA). None of the UK subjects had ever received AAT augmentation
therapy. A full clinical assessment including smoke exposure, presence of chronic
bronchitis (defined as a productive cough for at least 3 months in at least 2 consecutive
years [21]), lung function testing and high resolution CT scanning of the chest was undertaken,
as described previously [22]. The presence of emphysema was determined by the appearance of the scan and density
mask analysis of slices at the level of the aortic arch (representing the upper zone)
and the inferior pulmonary vein (representing the lower zone) using a threshold of
-910 Hounsfield Units (HU). This HU threshold has been validated against physiological
measures in AATD [22]. Patients whose voxel index exceeded values seen in normal subjects in either zone
[23] were classified as having emphysema.

Genotyping

Two SNPs (rs8034191 and rs1051730) in chromosome 15 were selected from the previous
GWA in COPD [15]. Additionally, 7 SNPs in IREB2 were selected using pairwise linkage disequilibrium (LD)-tagging in Tagger with a
minimum minor allele frequency of 0.05 and r2-threshold of 0.8 [24]. SNPs were genotyped using Sequenom (San Diego, CA) assays in the AAT Genetic Modifiers
Study. All family data were evaluated for familial inconsistencies using the PEDCHECK
program [25].

In UK AATD National Registry study, genotyping was carried out using TaqMan® technologies (Applied Biosystems, UK) on an ABI7900 HT for 3 SNPs associated in the
test dataset (rs2568494, IREB2; rs8034191, LOC123688; rs1051730, CHRNA3). All genotyping assays were pre-validated by the suppliers, and all plates included
appropriate negative controls.

Statistical analysis

Hardy-Weinberg equilibrium was assessed using goodness of fit tests. Pre- and post-bronchodilator
FEV1 percent of predicted and pre- and post-bronchodilator FEV1/FVC ratio were analyzed as quantitative phenotypes. Family-based association analysis
was performed using PBAT software version 3.6 [26] assuming additive genetic models, adjusting for pack-years and pack-years2 of cigarette smoking, under the null hypothesis of no linkage and no association in
the AAT Genetic Modifiers Study. In addition to the overall model, we evaluated gender-stratified
models and models that included a SNP-by-smoking or a SNP-by-gender interaction term.
Haplotype analysis was performed using 8, 4, 3, 2 SNP adjacent sliding windows in
PBAT.

In the UK AATD National Registry study, data were analyzed using SPSS (version 16,
Chicago: SPSS Inc). Quantitative genetic association analysis was carried out for
FEV1 and FEV1/FVC using general linear models, adjusting for age, gender and smoke exposure (as
pack-years and pack-years2). Logistic regression models were used for the presence of emphysema or COPD (defined
as post bronchodilator FEV1/FVC < 0.7) accounting for covariates as before. Additive models were assumed for
all SNPs. Gender stratification and SNP-by-gender and SNP-by-smoking interaction analyses
were also carried out, as in the test dataset.

Results

Demographic characteristics

The mean age of subjects was 52.2 years, and mean post-bronchodilator FEV1 was 65.9% predicted in the AAT Genetic Modifiers Study and 50.3 years and 53.8% predicted
in the UK AATD National Registry, respectively (Table 1). Male subjects were 46% in AAT Genetic Modifiers Study and 59% in UK AATD National
Registry, respectively. Three hundred and sixty-six subjects (79.8%) had emphysema
in UK AATD National Registry.

Table 1. Baseline characteristics for PI ZZ individuals in the AAT Genetic Modifiers Study
and the UK AATD National Registry

Association analysis

There were no deviations from Hardy Weinberg equilibrium for any of the genotyped
SNPs.

In the AAT Genetic Modifiers Study, three SNPs (rs2568494 in IREB2, rs8034191 in LOC123688, and rs1051730 in CHRNA3) were associated with pre-bronchodilator FEV1 percent of predicted (p < 0.05 Table 2 Figure 1). Two SNPs (rs2568494 and rs1051730) were associated with post-bronchodilator FEV1 percent of predicted and pre-bronchodilator FEV1/FVC ratio. One SNP (rs1051730) was associated with post-bronchodilator FEV1/FVC ratio (Table 2). Linkage disequilibrium (assessed with r2) between rs8034191 and the 1051730 was 0.9 (Figure 2). There was significant association only with a 2 SNP haplotype including rs8034191
and rs1051730 with pre- bronchodilator FEV1/FVC ratio (global test statistic; p = 0.05) using PBAT.

Interactions with cigarette smoking

There was no association between any of the genotyped SNPs and pack-years of smoking
as the outcome in the AAT Genetic Modifiers Study. Inclusion of a SNP-by-pack-years
interaction term for the lung function phenotypes showed significant interaction of
rs1051730 with pack-years of smoking for the pre- bronchodilator FEV1/FVC ratio (main effect; p = 0.02, interaction effect; p = 0.04). There was no significant association with lung function phenotypes when the
study population was stratified into groups of ever-smokers (n = 233) and never-smokers (n = 145) although this stratified analysis reduced the number of informative families
considerably.

In the stratified analysis, for the male subgroup, the p values were similar to the whole cohort results, with rs8034191 showing significant
association with pre- and post-bronchodilator FEV1 percent of predicted and post-bronchodilator FEV1/FVC ratio (Table 3). However, in the female subgroup, there was no significant association with lung
function phenotypes.

Replication analysis

In the initial analyses in the whole UK dataset, no significant associations with
quantitative phenotypes including FEV1 and FEV1/FVC and qualitative presence of emphysema and COPD were observed (all p > 0.05). Gender interaction was apparent for rs2568494 with both COPD and emphysema
(main effect p = 0.10, interaction p = 0.04 and 0.06, 0.03 respectively). No other statistically significant gender interactions
were observed. In the sex-stratified models, evidence of association for SNPs in IREB2 and LOC123688 was observed. A trend was observed for association of rs8034191 and rs2568494 with
COPD in the male subgroup, the risk alleles being C and A respectively (both p = 0.09). The SNP rs2568494 in IREB2 was significantly associated with emphysema in the male subgroup, the A allele conferring
an odds ratio and 95% confidence interval (OR and 95% CI) of disease of 2.67 (1.10-6.51,
p = 0.03). No association was seen with rs1051730 with emphysema.

With addition of a SNP-by-smoking interaction term, both rs8034191 and rs2568494 were
associated with COPD in the male subgroup (main effect, p = 0.03; interaction effect, p = 0.02; and main effect, p = 0.04; interaction effect, p = 0.003, respectively). Similar associations with emphysema were seen for rs2568494
(p = 0.03 and 0.02 respectively). Positive findings in the two datasets are summarized
in Table 4.

Table 4. Positive findings of genetic association analysis in the AAT Genetic Modifiers Study
and the UK AATD National Registry

Discussion

SNPs in the chromosome 15 CHRNA3/CHRNA5/LOC123688/IREB2 region have been shown to have associations with lung cancer and COPD unrelated to
AAT deficiency. In our current analysis, SNPs in IREB2, LOC123688 and CHRNA3 genes were shown to be associated with lung function phenotypes in AAT deficient subjects
(all PI ZZ) from the AAT Genetic Modifiers Study, and suggested a potential sex-specific
effect. Replication in another cohort of AAT deficient subjects from the UK showed
that a SNP in IREB2 was also associated with emphysema in men. This suggests that chromosome 15q region
genes that were found by GWA studies and gene expression analysis of lung tissue samples
may also be modifier genes of COPD and emphysema in AAT deficient subjects.

CHRNA3 was associated with lung cancer in three separate large GWA studies. This gene was
associated with COPD by GWA and the association was replicated in two other COPD cohorts.
There have also been recent reports of an association with smoking addiction [27], so it is unclear whether the lung cancer and COPD associations relate to smoking
behavior, another aspect of lung biology, or both. CHRNA3 is a subunit gene of the nicotinic cholinergic receptor and expressed in autonomic
ganglia and brain but is also expressed in bronchial and non-bronchial epithelial
cells [28]. Expression in lung cancer cells and signal transduction and apoptosis studies suggests
a potential role in carcinogenesis [29]. Interestingly, there are not many observations of lung cancer in patients with AAT
deficiency, perhaps because of mortality associated with the development of severe
COPD at an early age. Whether there is a common mechanism unrelated to smoking in
the pathogenesis of lung cancer and COPD, or whether these previously reported associations
relate to smoking addiction is unclear.

IREB2 is a protein of iron-responsive elements (IREs) and is regulated in response
to iron and oxygen supply [30]. IREB2-/- mice have aberrant iron homeostasis and accumulate iron in the intestine
and the central nervous system(CNS); the CNS accumulation may lead to neurodegenerative
disease [31]. Excess iron can be toxic, but the mechanism of neurodegenerative disease is unclear;
work is in progress to further characterize the functional pathways impacted by IREB2 in the lung. IREB2 was found to be differentially expressed according to lung function by microarray
experiments, and the SNPs in IREB2 showed associations in both a COPD case-control study and family-based studies including
the Boston Early-onset COPD and International COPD Genetics Network studies [18]. In a recent report, IREB2 polymorphisms were associated with COPD susceptibility in a European population [32]. Interestingly, rs2568494 was significantly associated with COPD in three studies
including our current study.

Previous studies of AAT deficient subjects showed that lung function was lower in
men than women [33], and previous analyses in the AAT Genetic Modifiers Study also showed lower lung
function in men [19]. Our current study suggests that genetic modifier effects of IREB2 and CHRNA3 may be more prominent in males--potentially contributing to some of the sex-specific
features of COPD susceptibility and severity among PI ZZ subjects, although a larger
sample size is needed to verify a gene-by-sex interaction.

In this study, there was no association between IREB2 and CHRNA3 genes and smoking intensity. In the AAT Genetic Modifiers Study, results showed no
association when the cohort was stratified by smoking history (ever smokers versus
never smokers). However, there was a marginal interaction of rs1051730 with smoking.
In the UK study, there were significant smoking interactions of rs2568494 and rs8034191.
Smoking markedly increases the risk of COPD and lowers the age-of-onset of COPD in
AAT deficient subjects [6,19], and despite small sample sizes, we found reasonable evidence for gene-by-smoking
interactions in the chromosome 15q region.

There are several limitations in this study. Multiple statistical comparisons are
a potential concern in any complex disease genetics study. Adjusting for either 3
genes or 9 SNPs tested, a p value of 0.02 is marginal. Additionally, the association with pulmonary function did
not replicate in the UK population, potentially due to phenotypic heterogeneity between
the two cohorts. Specifically, the UK subjects have lower mean FEV1 and potentially
more emphysema, both of which could influence non-replication. Of note, the association
with emphysema was investigated only in the UK population as chest CT scan data collection
was not part of the AAT Genetic Modifiers Study. Considering that these SNPs (rs2568494
in IREB2, rs8034191 in LOC123688, and rs1051730 in CHRNA3) were associated with intermediate phenotypes of COPD in other populations and that
we include an independent AAT deficient replication cohort, our result are likely
meaningful. Additionally, this test- replication approach is even more appealing since
all subjects were homozygous recessive for the AAT risk locus (PI ZZ). Also, replication
of our results showed association with emphysema, a less heterogeneous pulmonary phenotype.
The associated SNPs included two intronic (rs2568494, rs8034191) and one synonymous
exonic (rs1051730) SNP. The exonic SNP was not associated with COPD-related phenotypes
in the UK cohort. Another limitation of our current study is that rare functional
variants in this chromosome 15 region may be contributing to the role of these genes
in COPD; genome sequencing efforts in AAT deficient cohorts would be valuable to study
rare variant associations. Functional data for associated variants are currently lacking,
but many groups are pursuing functional work on this chromosome 15 region.

Conclusion

We have identified that the chromosome 15q25 region likely contains at least one potential
modifier gene of COPD phenotypes in individuals with severe AAT deficiency. The association
may be due to smoking behavior, but this is less likely; additionally, these associations
may have sex-specific effects. Future directions will include further evaluation of
the gene-by-sex interaction in larger cohort with AAT deficiency and identification
of the functional variant or variants in this region.

Abbreviations

Competing interests

WJK, AMW, AFB, MLB, EJC, EE, GM, RAS and GT have reported that no potential conflicts
of interest. SIR was supported from GlaxoSmithKline for travel to meetings for the
study. JMS received grant support from Talecris, Baxter. JKS received grant support
from AstraZeneca, and honoraria from Talecris, Baxter, CSL Behring, Boehringer Ingelheim,
Kamada, Grifols, and has received fees for participation in review activities from
Shire and AsthmaTx. CS received consulting fees from AstraZeneca, Talecris, Baxter,
Forest, Phamaceuticals, Uptake Medical, Pulmonx and payment for lectures from Talecris
and AstraZeneca. EKS received grant support and consulting fees from GlaxoSmithKline
for studies of COPD genetics and honoraria and consulting fees from AstraZeneca. RAS
received grant support, honoraria and consulting fees and supported for travel to
meetings for the study from Talecris. DLD received grant support from Doris Duke Charitable
Foundation.

Authors' contributions

All authors contributed to the study design, data collection and analysis, and writing
of the manuscript. WJK, AMW, DLD contributed to data analysis. All authors read and
approved the final manuscript.

Acknowledgements

This work was supported by US National Institutes of Health [Grant R01 HL68926, R01
HL075478, R01 HL084323, P01 HL083069 (EKS) and HL089438]. The UK AATD Registry was
supported by an unrestricted grant by Talecris as part of the ADAPT program. Additionally,
DLD is supported by a Clinician Scientist Development Award from the Doris Duke Foundation.